This application relates to single chain antibody constructs which specifically bind to the disialoganglioside GD2, and to the use of such constructs for targeted delivery of imaging agents or therapeutic agents to human neuroectodermal derived cancers.
Gangliosides are acidic glycosphingolipids found on the outer surface of most cell membranes.1 Many tumors have abnormal glycolipid composition and structure. Disialoganglioside GD2 has been found in a wide spectrum of human tumors, including neuroblastoma, osteosarcomas and other soft tissue sarcomas, medulloblastomas, high grade astrocytomas, melanomas, and small cell lung cancer.2-4 Among glioblastoma multiforme and anaplastic astrocytoma, anti-GD2 demonstrated the most restrictive pattern when compared with anti-GD3 and anti-GM2 antibodies.5.6 
Gangliosides are ideal targets for monoclonal antibodies (MAb) because of the high antigen density, lack of modulation, relative homogeneity in many tumors and the possibility of up-regulation by cytokines.7 The only normal tissues with high ganglioside expression are neurons, and biodistribution studies have shown that MAb do not localize to the nontumorous brain or spinal cord because of the blood brain barrier. In contrast, in patients with primary or metastatic brain tumors, specific antibodies can localize preferentially to tumor tissues, but not to normal brain.8 
Murine monoclonal antibodies have been prepared to ganglioside GD2. Using somatic cell hybridization, murine MAbs were produced against the ganglioside GD2.9 They were shown to react with disialoganglioside GD2, but not with GD3, GT1b, GD1b, GD1a, GM1, GM3 and GM4. When base-treatment step was omitted from the standard neuroblastoma ganglioside extraction procedure, immuno-thin-layer-chromatography (ITLC) using 3F8, 3G6 and other anti-GD2 MAbs revealed a new ganglioside band with Rf of 0.342, besides GD2 (Rf 0.183).4 Immunochemical analysis showed that this new neuroblastoma ganglioside contained alkali-sensitive O-acetylated sialic acid residues recognized by MAb D1.1.
Of 15 anti-GD2 MAbs studied, 13 reacted strongly with the novel ganglioside. 3F8 was chosen for our initial clinical studies because of its being an IgG3 and its strong binding in vitro to GD2. Based on the cDNA sequence and the anti-idiotype cross-reactivity, the antigen specificity and affinity of 3f8 and 3G6 were similar if not identical. We have chosen 3G6 for scFv development for ease of comparison with the other 14 MAbs which are IgM antibodies.
In order to determine the general applicability of the ganglioside GD2 as a target for immunotherapy, its expression in human cancers has been studied by immunostaining tumor specimens using these monoclonal antibodies. These anti-GD2 antibodies reacted with all the neuroblastoma surgical specimens tested to date in our laboratory. A recent update10 analyzed a series of 39 neuroblastomas. Staining of both primitive neuroblastic and differentiating ganglioneuromatous elements were seen, although tumor cell heterogeneity was noted in some. 23/39 tumors showed a more intense reactivity with MAb 3A7 than with 3F8, and this was particularly evident in the primitive neuroblastoma group. In a separate study, the expression of GD2 was analyzed in 67 solid tumors and normal tissues from children by using the antibody 3A7.11 GD2 expression was found in 28 of 28 neuroblastomas, and was most abundant in stroma-poor tumors. Differentiating stroma-rich neuroblastomas, neuroblastic clusters, neurofibrils, and most ganglion-like cells were found to be GD2 positive, whereas Schwann""s-cell stroma did not express GD2. In ganglioneuromas, only a few ganglion-like cells showed GD2, whereas all other structures were negative. Scattered foci of GD2 were also found in some non-neuronal tumors, such as rhabdomyosarcomas and osteosarcomas, but not in lymphomas, Askin tumors, or most Wilm""s tumors. 3A7 was also found to react with retinoblastomas.12 
Previous studies have shown that anti-GD2 antibodies reacted with the majority of osteosarcomas.13 Sixty freshly frozen human soft-tissue sarcomas were studied by avidinbiotin immunostaining using purified monoclonal antibodies 3F8 (anti-GD2) and R24 (anti-GD3).14 Ninety-three percent of the tumors tested by the immunohistochemical staining expressed GD2 and 88% expressed GD3. The intensity of expression varied among different histologic types. Liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma and spindle cell sarcoma reacted strongly with both antibodies. Embryonal rhabdomyosarcoma and synovial sarcoma demonstrated substantially weaker staining by either MAb. Ganglioside extraction and immuno-thin layer chromatography (ITLC) confirmed the identities of these gangliosides as GD2 and GD3 respectively.
Among brain tumors, 3F8 and 3A7 have also shown excellent reactivities. Two separate studies were carried out the first study in collaboration with Dr. Paul Zeltzer of Texas and the second with Dr. Ira Bergman (now Associate Professor of Neurology at the University of Pittsburgh) in our laboratory. In the first study, 12/15 medulloblastoma and 16/18 astrocytoma were positive, the majority staining homogeneously. In the second study, similar results were obtained. Medulloblastoma and a number of brain tumors reacted strongly with 3F8 and 3A7. The pattern of reactivity was generally homogeneous. For small cell lung cancer, all have reacted homogeneously in vitro using immunoperoxidase techniques.
Despite in vitro evidence for exquisite specificity of these antibodies for the ganglioside GD2 on neuroblastoma cells, a critical test of in vivo delivery is the actual amount of MAb uptake in the tumors. Biodistribution of 131I-anti-GD2 antibody was tested in preclinical experiments using athymic mice xenografted with human neuroblastoma.
Between 8 to 50% injected dose of 131I-MAb/gm of tumor was found, with variability depending primarily on the size of the tumor.15 There was no localization to GD2-negative tumors like Ewing""s sarcoma. Pooled mouse IgG and an irrelevant MAb also did not localize to neuroblastoma xenografts. Both small tumors (50 mg) and large tumors (over 2 g) showed radiolocalization with this technique. Optimal tumor to normal tissue ratios were rapidly reached by 24 to 48 hours. There was no increased uptake in the reticuloendothelial system, and the MAb did not cross the intact blood-brain barrier. The efficacy of tumor targeting was then tested by imaging neuroblastoma patients with 131I-MAb. Radiolocalization was demonstrated in primary tumors of the mediastinum and abdomen, as well as metastatic disease in the lymph nodes, bone marrow and bone.61.17 The specificity was validated by tumor and marrow biopsies, as well as by CT/MRI and bone scans. A comparison with 131I-meta-iodobenzylguanidine (MIBG) suggested that 131I-MAb was twice as sensitive in detecting metastatic sites of disease. The tumor uptake in patients was 0.08% of the injected dose per gm (compared to 0.002% for MIBG). This high tumor uptake in vivo was a result of (1) the high density (5xc3x97106/cell) and homogeneity of the target antigen GD2, and (2) the lack of uptake in the reticuloendothelial system. A number of human cancers has been imaged using GD2 specific antibodies. These include small cell lung cancer,18 brain tumors,8 and both osteosarcomas13 and soft tissue sarcomas.19 
A phase I study to test the biological toxicity of xe2x80x9ccoldxe2x80x9d anti-GD2 was carried out in 1987 in 17 patients with metastatic neuroblastoma or melanoma. A subsequent phase II study was carried out in 16 patients with stage IV neuroblastoma. Acute self-limited toxicities of MAb treatment were severe pain requiring analgesics, fever, urticaria, hypertension, hypotension, anaphylactoid reactions of the respiratory tract, as well as significant decreases in blood counts and serum complement levels. There were no treatment related deaths. Among the 5 neuroblastoma patients who are still alive and well (19 mos, 3 y, 5 y, 5 y, 6 y respectively after MAb treatment), there are no acute or delayed neurological complications attributable to MAb therapy. Among the survivors, one patient had chemo-radiotherapy-resistant stage IVS neuroblastoma, and the other 4 had poor risk stage IV neuroblastoma diagnosed at more than one year of age (2 relapsed neuroblastoma and 2 with refractory neuroblastoma prior to antibody treatment).
More recently, a phase I study to determine the radiological toxicity was carried out. Twenty-three patients (11 M and 21 F, ranging from 0.3 to 24.2 years of age at diagnosis) with refractory neuroblastoma (22 stage IV, 1 stage IIIU), were treated with 131I-3F8 at 7 dose levels, namely 6, 8, 12, 16, 20, 24, and 28 mCi/kg. Radiation dose to the blood was calculated based on blood clearance total body dose was based on total body clearance, and the tumor/organ dose on regions of interest calculations from serial gamma imagings. 21/23 patients were rescued with autologous bone marrow; one patient received GM-CSF alone; one died of progressive disease before marrow reinfusion. Marrow was infused when blood radioactivity decreased to  less than 0.01 uCi/ml in the first 18 patients and to  less than 1 uCi/ml in the last 4 patients. Acute toxicities of 131I-MAb treatment included pain (19/23) during the infusion, fever (19/23), hyperbilirubinemia (6/23), and diarrhea. All patients developed grade 4 myelosuppression with sepsis in 7/23 patients (5 fungal, 2 bacterial), disseminated zoster in 1, and pneumocystis in 1. Despite orally administered saturated solution of potassium iodide, 3 patients developed hypothyroidism. Subsequent 14 patients were treated with synthroid or Cytomel for thyroid protection. No other significant extramedullary toxicities have been encountered in patients followed past 20 months (50+, 40+, 30+, 26+, 23+, mos) from the time of 131I-MAb treatment. Fourteen patients have died, 11 of disease and 3 from infections during the cytopenic period, and in 4 patients follow-up is still short. Responses were seen in both soft tissue masses and bone marrow. Average tumor dose was 150 rad/mCi/kg. We concluded that when 131I-MAb was administered intravenously (6-28 mCi/kg), significant toxicities were encountered, including myelosuppression and their infectious complications, pain, fever, as well as hypothyroidism. Autologous marrow rescue could reverse marrow aplasia and thyroid supplement was essential to prevent thyroid damage. Although severe extramedullary toxicities were not seen, improvement in the pharmacokinetics of the radioconjugates will reduce significantly the marrow toxicity.
To date, a total of  greater than 95 patients have been treated with antibody 3F8, and more than 120 imaging studies have been carried out on different ongoing protocols. Among pediatric patients, no neuropathy has been reported, either sensory or motor in nature. More than two thirds of these patients mounted HAMA response, mostly low titer and not persistent. There was no correlation of HAMA with toxicity. Nevertheless, in view of the neuropathy seen with other anti-GD2 antibodies 14.2a and 14.18 (similar in reactivity patterns to 3F8), we want to improve the specificity to reduce side effects. All of these clinical trials have been carried out using antibodies produced at Memorial Sloan-Kettering Cancer Center using guidelines of the Office of Biologics Research and Review Center for Drugs and Biologics, Food and Drug Administration. For quality assurance, hybridoma 3F8 was found to be negative for adventitious agents by MAP, S+L-, and XC plaque assays, as well as negative for reverse transcriptase. MAP testing included screening for murine leukovirus, LCM virus isolation by intracerebral inoculation, murine saliva gland virus, mouse thymic virus, EDIM and LDH virus isolations. Purified antibody (e.g. 3F8) had to pass MAP and sterility testing (bacteria, mycoplasma, and fungal cultures), rabbit pyrogen testing, as well as safety testing in mice and guinea pigs. Conjugation to 131I by the chloramine-T method was supervised by Dr. Ronald Finn and Dr. Steven Larson in the Department of Nuclear Medicine. Specific activity of iodine-131 was  greater than 600 mCi/ug iodide. Radiolabeled antibody 3F8 must have  greater than 50% binding by in vitro antigen binding assay,  greater than 95% TCA precipitable and  less than 3% free iodine by radio-thin layer chromatography. Periodic testing of radiolabeled antibodies was performed to ensure sterility as well as the absence of pyrogen.
Although the wide expression of GD2 in human neuroectodermal-derived cancers (melanoma, small cell lung cancer, neuroblastoma, brain tumors, sarcoma, HTLV-1 leukemia, retinoblastoma and osteosarcoma) and the preliminary clinical studies of monoclonal antibodies to GD2 in radioimmuno-scintigraphy and radioimmunotherapy have been encouraging, further optimization of antibodies for binding to GD2 would be desirable. It is an object of the present invention to provide such optimized antibodies and the DNA sequences coding therefore.
It is a further object of the invention to provide methods of using the optimized antibodies and DNA sequences in diagnostic assays and therapeutic techniques.
The antibodies of the present invention are recombinant antibody constructs comprising the variable regions of the heavy and light chains of anti-GD2 antibodies. These antibody constructs may be coupled to a label such as a radiolabel or to a protein such as streptavidin or pro-drug converting enzymes for use in imaging or therapeutic applications. The antibody constructs may also be transduced into T cells to produce populations of T cells which target GD2-producing tumor cells.